Two prokaryotic systems will be employed to explore some of the important umresolved problems with respect to the energization of ATP synthesis. An obligately alkalophilic bacterium, Bacillus alcalophilus, and an uncoupler-resistant strain of Bacillus megaterium will be studied. Both of those organisms produce ATP via oxidative phosphorylation under conditions in which the protonmotive force is as low as -45mV. Thus the findings in these systems raise questions with respect to the chemiosmotic proposal which posits that ATP synthesis is completely coupled to the protonmotive force (the transmembrane electrochemical proton gradient) and that 2H+/ATP are translocated through the ATPase. In the two experimental systems, the relationship between the magnitude of the protonmotive force and phosphorylation potential will be characterized in detail. Proton translocation, concomitant with ATP synthesis, will also be studied as a function of the protonmotive force. H+/ATP stoichiometries will be measured. Energization be respiration-induced gradients will be compared to energization by artificial gradients, as an approach to the possible involvement of some "microscopic" gradient. Purification, characterization, and reconstitution experiments on the ATPases will be conducted to specifically identify structural and functional properties that may relate to the ability to synthesize ATP at low protonmotive forces. The studies of the uncoupler-resistant B. megaterium strain will be conducted in comparison with the wild type strain. The usefulness of prokaryotes in the exploration of basic bioenergetic problems is already established. The proposed experimental systems are mutable, subject to readily controlled growth conditions, and easily converted to vesicles. Since energy transduction is a fundamental biochemical process, it relates to the normal and pathological states of all living forms.